Azole peptidomimetics as thrombin receptor antagonists

ABSTRACT

Azole derivatives of formula (I): ##STR1## are disclosed as useful in treating platelet-mediated thrombotic disorders.

This is a provision of Ser. No. 60/075,171 filed Feb. 14, 1998 and adivision of application Ser. No. 09/245,739, filed Feb. 8, 1999 and nowU.S. Pat. No. 6,017,890.

BACKGROUND OF THE INVENTION

Thrombin is an important serine protease in hemostatis and thrombosis.One of the key actions of thrombin is receptor activation. A functionalhuman thrombin receptor, cloned by Coughlin in 1991 (T.-K. Vu, Cell1991, 64, 1057), was found to be a member of the G-protein coupledreceptor (GPCR) superfamily. The receptor activation putatively occursby N-terminal recognition and proteolytic cleavage at the Arg-41/Ser-42peptide bond to reveal a truncated N-terminus. This new receptorsequence, which has an SFLLRN [SEQ ID.:1] (Ser-Phe-Leu-Leu-Arg-Asn)N-terminus acting as a tethered ligand to recognize a site on thereceptor, can trigger activation and signal transduction leading toplatelet aggregation. Since 1991, two other protease-activated receptorswith extensive homology to the thrombin receptor, "PAR-2" (S. Nystedt,Proc. Natl. Acac. Sci USA 1994, 91, 9208) and "PAR-3" (H. Ishihara,Nature 1997, 386, 502), were cloned, and found to be activated bysimilar N-terminal hexapeptide sequences. Thrombin receptor (PAR-1)specific antibody-induced blockade of the platelet thrombin receptor hasshown efficacy against arterial thrombosis in vivo (J. J. CookCirculation 1995, 91, 2961). Hence, antagonists of the thrombin receptorbased on SFLLRN are useful in antagonizing these protease-activatedreceptors and as such may be used to treat platelet mediated thromboticdisorders such as myocardial infarction, stroke, restenosis, angina,atherosclerosis, and ischemic attacks by virtue of their ability toprevent platelet aggregation.

The thrombin receptor has also been identified on other cell types:endothelial, fibroblast, osteosarcoma, smooth muscle, and neuronal/glia.Thrombin activation of endothelial cells upregulates P-selectin toinduce polymorphonuclear leukocyte adhesion--an inflammatory response ofthe vessel wall (Y. Sugama, J. Cell Biol. 1992, 119, 935). Infibroblasts, thrombin receptor activation induces proliferation andtransmission of mitogenic signals (D. T. Hung, J. Cell Biol. 1992, 116,827). Thrombin has been implicated in osteoblast proliferation throughits activation of osteoblast cells (D. N. Tatakis, Biochem. Biophys.Res. Commun. 1991, 174, 181). Thrombin has been implicated in theregulation and retraction of neurons (K. Jalink, J. Cell. Biol. 1992,118, 411). Therefore, in this context the antogonist compounds of thisinvention may also be useful against inflammation, restenosis,osteoporosis, and neurodegenerative disorders.

The compounds of the present invention are azole peptidomimeticsrepresented by the general formula (I) below. Azole-containing cyclicpeptides have been synthesized to be employed as cytotoxic agents (C.Boden, Tetrahedron Lett. 1994, 35, 8271). By contrast, the azolepeptidomimetics of the present invention are strictly acyclic withactivity against the thrombin receptor. Azole-based dolastatin analogueshave been prepared as antitumor agents (K.Sakakibara, PCT Int. Appl., 31pp., WO9633212). These compounds contain a 4-thiazole-alkylamideC-terminus, whereas the compounds of the present invention require atleast two amino acid residues C-terminal to the 4-thiazole carboxamidefor activity against the thrombin receptor. Similarly, azole endothelinantagonists have been prepared which contain a 4-thiazole-carboxylicacid C-terminus (T. von Geldern, J. Med. Chem. 1996, 39, 957).

DISCLOSURE OF THE INVENTION

The present invention is directed to compounds represented by thefollowing general formula (I): ##STR2## wherein A1, A2, A3, A4, R1, R2,R3, and X are as hereinafter defined. The compounds of the presentinvention are platelet aggregation inhibitors and as such are useful intreating platelet-mediated thrombotic disorders such as arterial andvenous thrombosis, acute myocardial infarction, reocclusion followingthrombolytic therapy and angioplasty, inflammation, unstable angina, anda variety of vaso-occlusive disorders. These compounds are also usefulas antithrombotics in conjunction with fibrinolytic therapy (e.g., t-PAor streptokinase). Pharmaceutical compositions containing such compoundsas the active ingredient as well as methods of preparing the compoundsare also part of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

More particularly, the present invention is directed to compounds of thefollowing formula (I): ##STR3##

wherein A₁ is an amino acid residue selected from Sar, Gly, His, His(CH₂Ph), Ile Ser, Thr, β-Ala, or Ala. A₁ may also be a C₂ -C₆ -acyl groupsuch as, for example, acetyl, propionyl or butyryl or a C₁ -C₈ -alkylgroup such as, for example, methyl, ethyl, propyl or butyl;

wherein A₂ is an alkyl amino acid residue selected from Cha, Leu, Ile,Asp, and Glu or an amino alkyl amino acid residue such as Lys, His, Orn,homoArg and Arg;

wherein A₃ is an amino alkyl amino acid residue selected from Lys, His,Orn, Arg and homoArg;

wherein A₄ is an arylalkyl residue selected from Phe and Tyr or anaralkylamino group such as benzylamino or a phenethylamino group;

wherein R₁ is selected from H or alkyl;

wherein R₂ is an aryl, substituted aryl, heteroaryl, substitutedheteroraryl, aralkyl or substituted aralkyl group, however R₂ ispreferably aralkyl;

wherein R₃ is selected from H or alkyl;

wherein X is selected from S, O, or NR₄, wherein R₄ is selected from Hor alkyl;

and the pharmaceutically acceptable salts thereof.

As used herein, unless otherwise noted alkyl and alkoxy whether usedalone or as part of a substituent group, include straight and branchedchains having 1-8 carbons. For example, alkyl radicals include methyl,ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl,n-pentyl, 3-(2-methyl)butyl, 2-pentyl, 2-methylbutyl, neopentyl,n-hexyl, 2-hexyl, 2-methylpentyl and the like. Alkoxy radicals areoxygen ethers formed from the previously described straight or branchedchain alkyl groups. Acyl radicals are residues having 2-6 carbon atomsderived from an organic acid by removal of the hydroxyl group.

The terms "aryl", "heteroaryl", "substituted aryl" and "substitutedheteroaryl" as used herein alone or in combination with other termsindicates aromatic or heteroaromatic groups such as phenyl, naphthyl,pyridyl, thienyl, furanyl, or quinolinyl wherein the substituent is ahalo, alkyl, amino, nitro or alkoxy group. The term "aralkyl" means analkyl group substituted with an aryl group.

Unless otherwise indicated, the other substituent on the carbon to whichR₂ is attached to hydrogen.

The compounds of the present invention may also be present in the formof a pharmaceutically acceptable salt. The pharmaceutically acceptablesalt generally takes a form in which the basic nitrogen is protonatedwith an inorganic or organic acid. Representative organic or inorganicacids include hydrochloric, hydrobromic, hydriodic, perchloric, sulfuricnitric, phosphoric, acetic, propionic, glycolic, lactic, succinic,maleic, fumaric, malic, tartaric, citric, benzoic, mandelic,methanesulfonic, hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic,2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic,salicylic, saccharinic or trifluoroacetic.

Particularly preferred compounds of the present invention include thosecompounds shown in Table I, where the amino acids bear the "L" absoluteconfiguration unless denoted otherwise.

                  TABLE I                                                         ______________________________________                                          #STR4##                                                                       #-     R3     A1       A2    A3    A4       X                               ______________________________________                                         1   H      Sar        Cha   Arg   NHCH.sub.2 Ph                                                                          O                                    2 H β-Ala Cha Arg NHCH.sub.2 Ph O                                        3 H Sar Cha Arg NH(CH.sub.2).sub.2 Ph S                                       4 H Sar Cha hArg Phe-NH.sub.2 S                                               5 H IIe Cha Arg Phe-NH.sub.2 S                                                6 H Sar Lys Arg NH(CH.sub.2).sub.2 Ph S                                       7 Me Sar Cha Arg NHCH.sub.2 Ph O                                              8 Me His(CH.sub.2 Ph) Cha Arg NHCH.sub.2 Ph O                                 9 Me Ac Cha Arg NHCH.sub.2 Ph O                                              10 Me Me.sub.2 Cha Arg NHCH.sub.2 Ph O                                      ______________________________________                                    

The antagonists of the present invention may be prepared as shown inScheme AA. Protected oxazole intermediates (AA2) can be prepared in twosteps from the corresponding dipeptide (AA1) by Burgess Reagent-mediatedcyclization to the oxazoline and then oxidation with, for example,t-butyl peroxybenzoate to give the oxazole (AA2). Dipeptides such as AA1can be synthesized from the corresponding protected amino acids usingstandard solution-phase peptide coupling conditions utilizing EDC as theactivating agent, NMM as the base and DCM as the solvent. Standardpeptide methods are employed to complete the synthesis (e.g. compound1). Boc removal from AA2 utilizing an acid such as, for example, TFA orHCl and coupling with Boc-Sar-OSu affords AA3. The ester is thensaponified with a base such as, for example, lithium hydroxide or anyalkali metal or alkaline earth metal base and the carboxylic acidproduct is coupled with H-Cha-OMe to give AA4. Saponification of AA4 wita base, such as lithium hydroxide, for example, coupling withH-Arg(Pmc)-NHBn (EDC), and deprotection with TFA affords the product(1). The aforementioned Arg reagent, and other Arg amides in general,can be prepared in two steps from Fmoc-Arg(Pmc)-OH by EDC-mediatedcoupling with benzylamine and then Fmoc removal with 20% piperidine indioxane. Although the Scheme is used to illustrate the preparation ofthose compounds wherein R₂ is p-F-Ph, all of the compounds of thepresent invention can be prepared using the method illustrating inScheme AA by utilizing an appropriately substituted oxazole, thiazole orimidazole as the starting material. Intermediate azoles other thanoxazole AA2 can be prepared according to the methods exemplified inSchemes AB, AC, and AD. ##STR5##

The thiazole intermediate AB2 can be prepared in three steps from anamino acid residue (AB1) using Hantzsch cyclization methodology (SchemeAB). AB1 is converted to the corresponding thioamide using Lawesson'sReagent. The thioamide is then alkylated with ethyl 3-bromopyruvate, andthe product is cyclized with trifluoroacetic anhydride to give AB2.Those compounds of the present invention wherein X is S can be preparedfrom AB2 using standard peptide coupling procedures as exemplified inScheme AA. ##STR6##

The 5-methyl-oxazole intermediate AC2 can be prepared in two steps froma dipeptide (AC1) (Scheme AC). AC1 is converted to the correspondingmethyl ketone using the Dess-Martin reagent and the methyl ketone isthen cyclized with triphenylphosphine/iodine to give AC2. In the case ofcompound 10, the N,N-dimethyl-p-F-Phe derivative is prepared byreductive alkylation with, for example, formaldehyde/sodiumtriacetoxyborohydride following Boc removal with, for example, TFA fromAC2, and then the synthesis completed as shown in Scheme AA.

Those starting materials wherein X is NR₄ can be synthesized accordingto methods known to those skilled in the art. (S. K. Thompson, J. Med.Chem. 1994, 37, 3100). In this procedure, EDC-mediated coupling ofBoc-p-Phe-OH (AD1) with 4-amino-isoxazole followed by hydrogenation (H₂/Pd-C) and sodium hydroxide-mediated cyclization provides thecorresponding 2-substituted-imidazole-4-carboxaldehyde (AD2, Scheme AD).Oxidation of this aldehyde to the corresponding imidazole-4-carboxylicacid using standard methods (NAClO₂) and trimethylsilyldiazomethaneesterification provides the imidazole AD3. Those compounds of thepresent invention wherein X is NR₄ can be prepared from AD3 usingstandard peptide coupling procedures as exemplified in Scheme AA.Alkylation of the imidazole by generally known techniques produces thosecompounds of the invention wherein R₄ is alkyl. ##STR7##

To prepare the pharmaceutical compositions of this invention, one ormore compounds of formula (I) or salt thereof of the invention as theactive ingredient, is intimately admixed with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques, whichcarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral such asintramuscular. In preparing the compositions in oral dosage form, any ofthe usual pharmaceutical media may be employed. Thus, for liquid oralpreparations, such as, for example, suspensions, elixirs and solutions,suitable carriers and additives include water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like; for solidoral preparations such as, for example, powders, capsules, caplets,gelcaps and tablets, suitable carriers and additives include starches,sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like. Because of their ease inadministration, tablets and capsules represent the most advantageousoral dosage unit form, in which case solid pharmaceutical carriers areobviously employed. If desired, tablets may be sugar coated or entericcoated by standard techniques. For parenterals, the carrier will usuallycomprise sterile water, though other ingredients, for example, forpurposes such as aiding solubility or for preservation, may be included.Injectable suspensions may also be prepared, in which case appropriateliquid carriers, suspending agents and the like may be employed. Thepharmaceutical compositions herein will contain, per dosage unit, e.g.,tablet, capsule, powder, injection, teaspoonful and the like, an amountof the active ingredient necessary to deliver an effective dose asdescribed above. The pharmaceutical compositions herein will contain,per unit dosage unit, e.g., tablet, capsule, powder, injection,suppository, teaspoonful and the like, of from about 0.03 mg to 100mg/kg (preferred 0.1-30 mg/kg) and may be given at a dosage of fromabout 0.1-300 mg/kg/day (preferred 1-50 mg/kg/day). The dosages,however, may be varied depending upon the requirement of the patients,the severity of the condition being treated and the compound beingemployed. The use of either daily administration or post-periodic dosingmay be employed.

BIOLOGY

The compounds of the present invention interrupt platelet activationinduced by thrombin's proteolytic cleavage of its platelet surfacereceptor, and thereby inhibit platelet aggregation. Such compounds are,therefore, useful in treating platelet-mediated thrombotic disorderssuch as arterial and venous thrombosis, acute myocardial infarction,reocclusion following thrombolytic therapy and angioplasty, and avariety of vaso-occlusive disorders.

IN VITRO THROMBIN RECEPTOR BINDING ASSAY

CHRF membranes (Jones, Biochim. Biophys. Acta 1992, 1136, 272) arethawed from -70° C., centrifuged at maximum speed for 5 min, washedtwice with binding buffer (50 mM HEPES containing 5 mM MgCl₂ and 0.1%BSA), and re-suspended in binding buffer (25 μg/100 mL). 100 μl ofmembranes are added to the 24-Wallac plates and delivered to the Tomtechapparatus. In a typical experiment, 6 μl of samples (from a 125 μg/mLintermediary plate, 20%DMSO) and 44 μl buffer are delivered to theplates (final conc. of compounds is 3.7 μg/mL, 0.6% DMSO). Similarly, 6μl 20%DMSO and 44 μl buffer are delivered to both column 1 (NSB) andcolumn 12 (TB). 10 μl Ser-pFPhe-Har-Leu-Har-Lys-Tyr-NH₂ [SEQ ID.:2](721-40; 500 μM in deionized water) is added to column 1. 50 μltritiated 721-40 (specific activity 46 Ci/mmol) is added to all thewells. The plates are mixed well for 20 seconds, incubated for 30 min,and then harvested with 10 mM HEPES/138 mM NaCl using the Skatronharvester. The filters (GF/C Brandel FPXLR 296) are presoaked 3 h in0.5% polyethyleneimine in HEPES/0.1 M N-acetylglucosamine) are set insaran wrap and dried for 3 min in the microwave, and placed in samplebags (Wallac 1450-432). 4.5 mL scintillation fluid (Wallac, BetaplateScint 1205-440) is added. The bags are sealed, placed in filtercassettes (Wallac 1450-104), and analyzed on the microbeta counter.

IN VITRO INHIBITION OF THROMBIN-INDUCED GEL-FILTERED PLATELETAGGREGATION ASSAY

The percentage of platelet aggregation is calculated as an increase inlight transmission of compound-treated platelet concentrate vs.control-treated platelet concentrate. Human blood is obtained from drugfree, normal donors in tubes containing 0.13 M sodium citrate. Plateletrich plasma (PRP) is collected by centrifugation of whole blood at 200×gfor 10 min at 25° C. The PRP (5 mL) is gel filtered through Sepharose 2B(bed volume 50 mL), and the platelet count is adjusted to 2×10⁷platelets per sample. The following constituents are added to asiliconized cuvette: concentrated platelet filtrate and Tyrode's buffer(0.14 M NaCl, 0.0027 M KCl, 0.012 M NaHCO₃, 0.76 mM Na₂ HPO4, 0.0055 Mglucose, 2 mg/mL BSA and 5.0 mM HEPES @ pH 7.4) in an amount equal to350 μl, 50 μl of 20 mM calcium and 50 μl of the test compound.Aggregation is monitored in a BIODATA aggregometer for the 3 minfollowing the addition of agonist (thrombin 50 μl of 1 unit/mL).

Table II shows the biological activity of the compounds of the presentinvention. The table contains IC₅₀ values (μM) of the compounds in athrombin receptor binding assay, and IC₅₀ values (μM) against plateletaggregation stimulated by two agonists, thrombin or SFLLRN-NH₂ [SEQID.:1] (TRAP).

                  TABLE II                                                        ______________________________________                                        Biological Activity                                                                    Thr Receptor Binding                                                                         Platelet Aggregation**                                Compound IC.sub.50 *    IC.sub.50 Thr*                                                                          IC.sub.50 TRAP*                             ______________________________________                                        1        2.0            25        10                                            2 8.2 10 7                                                                    3 35.0 12 0.6                                                                 4 5.0 45 3                                                                    5 3.5 43 9                                                                    6 15.5 26 4                                                                   7 7.0 19 22                                                                   8 30.0 13 5                                                                   9 31.0 24 11                                                                  10 NT 11 17                                                                 ______________________________________                                         *μM                                                                        **Thrombininduced aggregation of gelfiltered platelets in μM.         

EXAMPLES

Protected amino acids were purchased from Fluka Chemical or BachemBioscience Inc. All other chemicals were purchased from Aldrich ChemicalCompany, Inc. High field ¹ H NMR spectra were recorded on a BrukerAC-360 spectrometer at 360 MHz, and coupling constants are given inHerz. Melting points were determined on a Mel-Temp II melting pointapparatus and are uncorrected. Microanalyses were performed at RobertsonMicrolit Laboratories, Inc., Madison, N.J.

In the examples and throughout this application, the followingabbreviations have the meanings recited hereinafter:

    ______________________________________                                        Ac          Acetyl                                                              Bn Benzyl                                                                     Boc t-Butoxycarbonyl                                                          Cbz Benzyloxycarbonyl                                                         CP compound                                                                   DCE 1,2-Dichloroethane                                                        DCM Dichloromethane                                                           DIC Diisopropylcarbodiimide                                                   DIEA Diisopropylethylamine                                                    DMAP 4-Dimethylaminopyridine                                                  DME 1,2-Dimethoxyethane                                                       DMF N,N-Dimethylformamide                                                     EDC Ethyl dimethylaminopropylcarbodiimide                                     EDTA Ethylenediaminetetraacetic acid                                          Et.sub.2 O Diethyl ether                                                      Fmoc 9-Fluorenylmethoxycarbonyl                                               HOBT Hydroxybenzotriazole                                                     i-Pr Isopropyl                                                                NMM N-Methylmorpholine                                                        OSu N-Oxysuccinimide                                                          Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl                                   PTSA p-Toluenesulfonic acid                                                   RT room temperature                                                           TFA Trifluoroacetic acid                                                    Amino acid abbreviations are defined below:                                        Ala        Alanine                                                         β-Ala beta-Alanine                                                       Arg Arginine                                                                  Asp Aspartic Acid                                                             Cha Cyclohexylalanine                                                         ρ-F-Phe 4-Fluorophenylalanine                                             Glu Glutamic Acid                                                             Gly Glycine                                                                   hArg Homoarginine (homoArg)                                                   His Histidine                                                                 Ile Isoleucine                                                                Leu Leucine                                                                   Lys Lysine                                                                    Or Ornithine                                                                  Phe Phenylalanine                                                             Sar Sarcosine                                                                 Ser Serine                                                                    Thr Threonine                                                                 Tyr Tyrosine                                                                ______________________________________                                    

Methyl2-[1(S)-t-butoxycarbonylamino-2-(4-fluorophenyl)ethyl]oxazole-4-carboxylate(AA2)

To a solution of Boc-p-F-Phe-OH (0.018 mol), DCM (200 mL), H-Ser-OMe(0.018 mol), HOBT (10 mg), and EDC.HCl (0.036 mol) at 5° C. was addedNMM (0.036 mol). The reaction was stirred for 3.5 h, diluted with sat'dNH₄ Cl (30 mL). The layers were separated, and the organic layer waswashed with sat'd NAHCO₃ (30 mL), dried (Na₂ SO₄), and evaporated togive a white powder (5.9 g). The powder was dissolved in DME (100 mL),treated with (methoxycarbonylsulfamoyl) triethylammonium hydroxide(0.015 mol), and heated at reflux for 1 h. The reaction was cooled toRT, diluted with EtOAc (150 mL) and sat'd NaHCO₃ (30 mL), and the layersseparated. The organic layer was dried (Na₂ SO₄) and evaporated to givea white solid (4.8 g). The solid was dissolved in benzene (140 mL),treated with Cu(OAc)₂ (0.014 mol), CuBr (0.014 mol), and t-butylperoxybenzoate (0.020 mol), and heated at reflux for 5 h. The reactionwas cooled, diluted with EtOAc (50 mL) and sat'd NaHCO₃ (10 mL), andfiltered. The layers of the filtrate were separated, and the organiclayer dried and evaporated to a brown oil. The oil was purified oversilica gel (2% MeOH/DCM) to give AA2 as a gold solid (1.75 g): ¹ H NMR(CDCl₃) δ8.12 (s, 1H), 7.6 (m, 1H), 6.9 (m, 4H), 5.2 (m, 1H), 3.91 (s,3H), 3.2 (m, 2H), 1.40 (s, 9H); FAB-MS m/e 365 (MH+).

The following examples describe the invention in greater detail and areintended to illustrate the invention but not to limit it.

Example 12-[1(S)-Sarcosineamido-2-(4-fluorophenyl)ethyl]oxazole-4-carboxycyclohexylalanyl-argininebenzylamide (1)

Intermediate AA2 (4.1 mmol) was dissolved in DCM (10 mL) and TFA (12 mL)and stirred for 1 h. The solution was concentrated to give a brown oil,and the oil triturated with hexane (50 mL). The oil was dissolved in DCM(60 mL), treated with Boc-Sar-OSu (4.1 mmol) and NMM (12.3 mmol), andstirred for 24 h. The reaction mixture was diluted with sat'd NH₄ Cl (15mL) and the layers separated. The organic layer was washed with sat'dNaHCO₃ (20 mL), dried Na₂ SO₄), evaporated, and purified by silica gelchromatography (2%MeOH/DCM) to give a brown oil (AA3, 1.0 g). AA3 (2.1mmol) was dissolved in THF (10 mL), cooled to 5° C., treated with aq.LiOH (4 mmol/20 mL water), and stirred for 2 h. The reaction wasacidified with citric acid (0.5 g) and extracted with CHCl₃ (2×70 mL).The organic materials were dried (Na₂ SO₄) and evaporated to give a goldfoam (0.85 g). The foam (2.0 mmol) was dissolved in DCM (60 mL) andtreated with H-CHa-OMe.HCl (2.0 mmol), HOBT (10 mg), EDC.HCl (3.0 mmol),and NMM (4.0 mmol). This mixture was stirred for 2 h, diluted with sat'dNH₄ Cl (20 mL), and the layers separated. The organic layer was dried,evaporated, and purified by silica gel chromatography (3% MeOH/DCM) toafford a gold oil (AA4, 1.0 g; FAB-MS m/e 589, MH+). AA4 (1.7 mmol) wasdissolved in THF (10 mL), cooled to 5° C., treated with aq. LiOH (3.4mmol/20 mL water), and stirred for 2 h. The reaction was acidified withcitric acid (0.5 g) and extracted with CHCl₃ (2×70 mL). The organicmaterials were dried (Na₂ SO₄) and evaporated to give a gold oil (0.71g). The oil (1.2 mmol) was dissolved in DCM (60 mL) and treated withH-Arg(Pmc)-NHCH₂ Ph (1.2 mmol), HOBT (10 mg), EDC.HCl (2.4 mmol), andNMM (1.2 mmol). This mixture was stirred for 2 h, diluted with sat'd NH₄Cl (20 mL), and the layers separated. The organic layer was dried (Na₂SO₄), evaporated, and purified by silica gel chromatography (7%EtOH/DCM) to afford a clear glass (0.90 g). The glass was dissolved inDCM (5 mL) and anisole (0.5 mL), treated with TFA (10 mL), and stirredfor 2.5 h. The solution was evaporated, and the resultant green oiltriturated with Et₂ O (4×30 mL), dried Na₂ SO₄), and isolated as a whitepowder (0.90 g): mp 127-130° C.; FAB-MS m/e 720 (MH⁺); [α]²⁴ D -21.8° (c0.28, MeOH). Anal. calcd. for C₃₇ H₅₀ N₉ O₅ F.2.0 TFA (947.91): C,51.95; H, 5.53; N, 13.30. Found: C, 51.53; H, 5.78; N, 13.05.

Example 22-[1(S)-β-Alanineamido-2-(4-fluorophenyl)ethyl]oxazole-4-carboxycyclohexylalanyl-argininebenzylamide (2)

Compound 2 was prepared using the method described in example 1.Intermediate AA2 (2.2 mmol) was deprotected with TFA and then reactedwith H-β-Ala-OSu (2.2 mmol) as described. Compound 2 was isolated as awhite powder (0.21 g); mp 108-112° C.; FAB-MS m/e 720 (MH+). Anal.calcd. for C₃₇ H₅₀ N₉ O₅ F.2.0 TFA.0.5 H₂ O (956.93): C, 51.46; H, 5.58;N, 13.17; KF, 0.91. Found: C, 51.41; H, 5.95; N, 13.20; KF, 0.88.

Ethyl2-[1(S)-t-butoxycarbonylamino-2-(4-fluorophenyl)ethyl]thiazole-4-carboxylate(AB2)

To a solution of AB1 (14.9 mmol) in dioxane (60 mL) was added Lawesson'sreagent (8.9 mmol). This mixture was stirred for 3 h, filtered, and thefiltrate evaporated and purified by silica gel chromatography (2%MeOH/DCM) to afford the thioamide (3.9 g). The thioamide (13.1 mmol) wasdissolved in DME (80 mL), treated with NaHCO₃ (0.10 mol) and ethylbromopyruvate (39.3 mmol), and stirred for 20 min. The mixture wascooled to 5° C., treated with a solution of TFAA (52.4 mmol), pyridine(0.10 mol), and DME (10 mL), and the ice bath removed. This mixture wasstirred for 17 h, filtered, concentrated, diluted with DCM, and washedwith water. The organic layer was dried (Na₂ SO₄) and purified by silicagel chromatography (1.5% MeOH/DCM) to afford AB2 as a white foam (4.6g); ¹ H NMR (CDCl₃) δ8.08 (s, 1H), 7.1 (m, 2H), 6.9 (m, 2H), 5.3 (m,2H), 4.4 (q, 2H), 3.2 (m, 2H), 1.5 (t, 3H), 1.40 (s, 9H); FAB-MS m/e 395(MH⁺).

Example 32-[1(S)-Sarcosineamido-2-(4-fluorophenyl)ethyl]thiazole-4-carboxy-cyclohexylalanyl-argininephenethylamide (3)

Compound 3 was prepared by the method described for compound 1.Intermediate AB1 (1.2 mmol) was dissolved in DCM (10 mL) and TFA (12 mL)and the resultant solution was stirred for 1 h. The solution was thenconcentrated to give a brown oil, and the oil triturated with hexane (50mL). The oil was dissolved in DCM (60 mL), treated with Boc-Sar-OSu (1mmol) and NMM (3 mmol), and stirred for 24 h. The reaction was dilutedwith sat'd NH₄ Cl (15 mL) and the layers separated. The organic layerwas washed with sat'd NaHCO₃ (20 mL), dried (Na₂ SO₄), evaporated, andpurified by silica gel chromatography (2%MeOH/DCM) to give an oil. Theoil was dissolved in THF (10 mL), cooled to 5° C., treated with aq. LiOH(1 mmol/6 mL water), and stirred for 2 h. The reaction was acidifiedwith citric acid (0.2 g) and extracted with CHCl₃ (2×70 mL). The organicmaterials were dried (Na₂ SO₄) and evaporated to give a foam. The foam(0.5 mmol) was dissolved in DCM (60 mL) and treated with H-Cha-OMe.HCl(0.5 mmol), HOBT (3 mg), EDC.HCl (1 mmol), and NMM (1.5 mmol). Thismixture was stirred for 2 h, diluted with sat'd NH₄ Cl (20 mL), and thelayers separated. The organic layer was dried (Na₂ SO₄), evaporated, andpurified by silica gel chromatography (3% MeOH/DCM) to afford a clearoil. The oil was dissolved in THF (5 mL), cooled to 5° C., treated withaq. LiOH (0.2 mmol/4 mL water), and stirred for 2 h. The reaction wasacidified with citric acid (0.5 g) and extracted with CHCl₃ (2×50 mL).The organics were dried (Na₂ SO₄) and evaporated to give a gold oil(0.71 g). The oil (1.2 mmol) was dissolved in DCM (60 mL) and treatedwith H-Arg(Pmc-NHCH_(CH) ₂ Ph (1.2 mmol), HOBT (10 mg), EDC.HCl (2.4mmol), and NMM (1.2 mmol). This mixture was stirred for 2 h, dilutedwith sat'd NH₄ Cl (20 mL), and the layers separated. The organic layerwas dried, evaporated, and purified by silica gel chromatography (7%EtOH/DCM) to afford a clear glass (0.3 g). The glass was dissolved inDCM (5 mL) and anisole (0.5 mL), treated with TFA (10 mL), and stirredfor 2.5 h. The solution was evaporated, and the resultant brown oiltriturated with Et₂ O (4×30 mL), dried and isolated as a beige powder(0.093 g): ¹ H NMR (DMSO-d₆) δ9.2 (m, 2H), 8.4 (d, 1H), 8.33 (s, 1H),8.2 (m, 1H), 8.1 (m, 1H), 7.6 (m, 1H), 6.9-7.4 (m, 9H), 5.4 (m, 1H), 4.6(m, 1H), 4.2 (m, 1H), 3.6 (q, 2H), 3.0-3.5 (m, 6H), 2.7 (m, 2H), 2.5 (m,2H), 2.39 (s, 3H), 1.3-1.9 (m, 10H), 0.8-1.3 (m, 10H); FAB-MS m/e 750(MH⁺).

Example 42-[1(S)-Sarcosineamido-2-(4-fluorophenyl)ethyl]thiazole-4-carboxy-cyclohexylalanyl-homoarginyl-phenylalanineamide(4)

Compound 4 was prepared by the method described in example 3 from AB2(1.0 mmol) and Boc-Sar-OSu (1.0 mmol), and isolated as a tan powder(0.021 g): ¹ H NMR (DMSO-d₆) δ8.38 (s, 1H), 8.0 (d, 1H), 7.9 (d, 1H),7.5 (m, 1H), 7.0-7.4 (m, 9H), 5.4 (m, 1H), 4.6 (m, 1H), 4.4 (m, 1H), 4.2(m, 1H), 3.7 (q, 2H), 3.4 (m, 4H), 3.1 (m, 4H), 2.8 (m, 2H), 2.47 (s,3H), 1.7 (m, 1H), 1.3-1.8 (m, 8H), 0.8-1.4 (m, 16H); FAB-MS m/e 807(MH⁺).

Example 52-[1(S)-Isoleucineamido-2-(4-fluorophenyl)ethyl]thiazole-4-carboxy-cyclohexylalanyl-arginyl-phenylalanineamide(5)

Compound 5 was prepared aby the method described in example 3 from AB2(1.3 mmol) and Boc-Ile-OH (1.3 mmol), and isolated as a pale yellowpowder (0.079 g): ¹ H NMR (DMSO-d₆) δ9.2 (m, 1H), 8.8 (m, 1H), 8.72 (s,1H), 7.7-8.1 (m, 7H), 7.6 (m, 1H), 6.8-7.5 (m, 9H), 5.4 (m, 1H), 4.6 (m,1H), 4.4 (m, 1H), 4.3 (m, 1H), 3.7 (m, 2H), 3.6 (m, 1H), 3.4 (m, 2H),2.7-3.2 (m, 6H), 1.8 (m, 2H), 1.0-1.7 (m, 18H), 0.9 (d, 3H), 0.8 (t,3H); FAB-MS m/e 835 (MH⁺).

Example 62-[1(S)-Sarcosineamido-2-(4-fluorophenyl)ethyl]thiazole-4-carboxy-lysyl-argininephenethylamide (6)

Compound 6 was prepared by the method described in example 3 from AB2(1.4 mmol) and Boc-Sar-OSu (1.4 mmol), and isolated as a tan powder(0.099 g): FAB-MS m/e 725 (MH⁺).

Methyl2-[1(S)-t-butoxycarbonylamino-2-(4-fluorophenyl)ethyl]-5-methyloxazole-4-carboxylate(AC2)

Dipeptide AC1 (12.6 mmol), prepared by the method described for thepreparation of Boc-p-F-Phe-Ser-OMe in example AA2, was dissolved in DCM(125 mL) and water (0.2 mL) and treated with1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benzodioxol-3-(1H)-one(Dess-Martin reagent; 15.1 mmol). The reaction was stirred for 30 min,diluted with DCM (100 mL), and washed with sat'd NaHCO₃ (2×40 mL),dried, and evaporated. The residue was purified by silica gelchromotography (30% EtOAc/hexane) to give a ketone. A solution of DCM(70 mL), PPh₃ (8.3 mmol), and TEA (16.5 mmol) was treated with theketone (8.3 mmol) and stirred for 5 min. The mixture was washed with aq.Na₂ S₂ O₃ and sat'd NaHCO₃, and the organic layer dried, evaporated, andpurified by silica gel chromatography (25% EtOAc/hexane) to give AC2 asa white foam (2.5 g): ¹ H NMR (CDCl₃) δ7.1 (m, 2H), 6.9 (m, 2H), 5.1 (m,2H), 3.92 (s, 3H), 3.2 (m, 2H), 2.60 (s, 3H), 1.40 (s, 9H); FAB-MS m/e379 (MH⁺).

Example 72-[1(S)-Sarcosineamido-2-(4-fluorophenyl)ethyl]-5-methyloxazole-4-carboxy-cyclohexylalanyl-argininebenzylamide (7)

Compound 7 was prepared by the method described for the preparation ofcompound 1. Intermediate AC1 (1.3 mmol) was dissolved in DCM (10 mL) andTFA (12 mL) and the solution was stirred for 1 h. The solution wasconcentrated to give a tan oil, and the oil triturated with hexane (50mL). The oil was dissolved in DCM (60 mL), treated with Boc-Sar-OSu (1.3mmol) and NMM (4 mmol), and stirred for 24 h. The reaction was dilutedwith sat'd NH₄ Cl (15 mL) and the layers separated. The organic layerwas washed with sat'd NaHCO₃ (20 mL), dried (Na₂ SO₄), evaporated, andpurified by silica gel chromatography (2%MeOH/DCM) to give an oil. Theoil was dissolved in THF (10 mL), cooled to 5° C., treated with aq. LiOH(1 mmol/6 mL water), and the mixture stirred for 2 h. The reactionmixture was acidified with citric acid (0.2 g) and extracted with CHCl₃(2×70 mL). The organic materials were dried (Na₂ SO₄) and evaporated togive a foam. The foam (0.6 mmol) was dissolved in DCM (60 mL) and thesolution was treated with H-Cha-OMe.HCl (0.6 mmol), HOBT (3 mg), EDC.HCl(1 mmol), and NMM (1.5 mmol). This mixture was stirred for 2 h, dilutedwith sat'd NH₄ Cl (15 mL), and the layers separated. The organic layerwas dried (Na₂ SO₄), evaporated, and the residue was purified by silicagel chromatography (3% MeOH/DCM) to afford a tan oil. The oil wasdissolved in THF (5 mL), cooled to 5° C., treated with aq. LiOH (0.2mmol/4 mL water), and the mixture was stirred for 2 h. The reactionmixture as then was acidified with citric acid (0.5 g) and extractedwith CHCl₃ (2×50 mL). The organic materials were dried (Na₂ SO₄) andevaporated to give a glass (0.71 g). The glass (1.2 mmol) was dissolvedin DCM (60 mL) and the solution was treated with H-Arg(Pmc)-NHBn (1.2mmol), HOBT (5 mg), EDC.HCl (2.4 mmol), and NMM (1.2 mmol). This mixturewas stirred for 2 h, diluted with sat'd NH₄ Cl (20 mL), and the layersseparated. The organic layer was dried (Na₂ SO₄), evaporated, and theresidue was purified by silica gel chromatography (7% EtOH/DCM) toafford a clear glass (0.3 g). The glass was dissolved in DCM (5 mL) andanisole (0.5 mL), the resultant solution was treated with TFA (10 mL),and stirred for 2.5 h. The solution was evaporated, and the resultantglass triturated with Et₂ O (4×25 mL), dried (Na₂ SO₄), and isolated asa white powder (0.10 g): mp 117-121° C.; FAB-MS m/e 734 (MH⁺). Anal.calcd. for C₃₈ H₅₂ N₉ O₅ F.2.0 TFA.0.5 H₂ O (970.94); C, 51.96; H, 5.71;N, 12.98; KF, 0.94. Found: C, 51.88; H, 5.89; N, 12.60; KF, 1.0.

Example 82-[1(S)-N(tau)-Benzyl-histidineamido-2-(4-fluorophenyl)ethyl]-5-methyloxazole-4-carboxy-cyclohexylalanyl-argininebenzylamide (8)

Compound 8 was prepared by the method described for the preparation ofcompound 7 from AC2 (1.1 mmol) and Boc-His(Bn)-OH (1.1 mmol), andisolated as a white powder (0.083 g): mp 101-106° C.; FAB-MS m/e 890(MH⁺). Anal. calcd. for C₄₈ H₆₀ N₁₁ O₅ F.2.6 TFA.0.8 anisole.1.0 H₂ O(1294.5): C, 54.61; H, 5.53; N, 11.90; KF, 1.39. Found: c, 54.23; H,5.44; N, 11.96; KF, 1.75.

Example 92-[1(S)-Acetamido-2-(4-fluorophenylethyl]-5-methyloxazole-4-carboxy-cyclohexylalanyl-argininebenzylamide (9)

Compound 9 was prepared by the method described for the preparation ofcompound 7 from AC2 (1.2 mmol) and acetyl chloride (1.2 mmol), andisolated as a white powder (0.10 g): mp 111-116° C.,; FAB-MS m/e 705(MH⁺). Anal. calcd. for C₃₇ H₄₉ N₈ O₅ F.1.0 TFA.1.0 anisole.1.0 H₂ O(901.78): C, 57.54; H, 6.35; N, 12.43; KF, 2.0. Found: C, 57.71; H,6.27; N, 12.49; KF, 2.32.

Example 102-[1(S)-N,N-Dimethyl-2-(4-fluorophenylethyl]-5-methyloxazole-4-carboxy-cyclohexylalanyl-argininebenzylamide (10)

Compound 10 was prepared by the method described for the peparation ofcompound 7, was synthesized via methyl2-[1(S)-N,N-dimethyl-2-(4-fluorophenyl)ethyl]-5-methyloxazole-4-carboxylateas follows. Intermediate AC2 (1.0 mmol) was deprotected of the Boc groupwith TFA as described. The primary amine TFA salt was partitionedbetween DCM (50 mL) and sat'd NaHCO₃ (15 mL), and the layers separated.The organic layer was dried (Na₂ SO₄) and evaporated to a glass. Theglass was dissolved in DCE (10 mL) and 37% formaldehyde (0.23 mL) at RTand then treated with sodium triacetoxyborohydride (4.0 mmol). Themixture was stirred for 18 h, diluted with DCM (50 mL), and washed withsat'd NAHCO₃ (10 mL). The organic layer was dried (Na₂ SO₄), evaporated,and the resultant foam purified by silica gel chromatography (1.5%MeOH/DCM) to afford methyl2-[1(S)-N,N-dimethyl-2-(4-fluorophenyl)ethyl]-5-methyloxazole-4-carboxylateas a glass (0.24 g). This glass was used to prepare 10 aby the methoddescribed in example 7. Compound 10 was isolated as a tan powder (0.088g): mp 115° C.; FAB-MS m/e 691 (MH⁺). Anal. calcd. for C₃₇ H₅₁ N₈ O₄F.2.0 TFA.2.1 H₂ O (956.75); C, 51.47; H, 6.03; N, 11.71; KF, 3.95.Found: C, 51.24; H, 6.28; N, 12.09; KF, 4.23.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 2                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:synthetic      - - <400> SEQUENCE: 1                                                         - - Ser Phe Leu Leu Arg Asn                                                    1               5                                                            - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:synthetic       - - <400> SEQUENCE: 2                                                         - - Ser Phe Arg Leu Arg Lys Tyr                                                1               5                                                          __________________________________________________________________________

We claim:
 1. A method of treating platelet-mediated thrombotic disorderscomprising administering to a patient afflicted with such disorder aneffective amount of the compound of the general formula (I) ##STR8##wherein A₁ is an amino acid residue selected from the Sar, Gly, His,His(CH₂ Ph), Ile, Ser, Thr, β-Ala, Ala, a C₂ -C₆ -acyl group and a C₁-C₈ -alkyl group;wherein A₂ is an alkyl amino acid residue selected fromCha, Leu, Ile, Asp, and Glu or an amino alkyl amino acid residueselected from Lys, His, Orn, homoArg and Arg; wherein A₃ is an aminoalkyl amino acid residue selected from Lys, His, Orn, Arg and homoArg;wherein A₄ is an arylalkyl residue selected from Phe and Tyr or anaralkylamino group; wherein R₁ is selected from H or alkyl; wherein R₂is an aryl, substituted aryl, heteroaryl, substituted heterorarylaralkyl or substituted aralkyl; wherein R₃ is H or alkyl; wherein X isselected from S, O, or NR₄, wherein R₄ is selected from H or alkyl; andthe pharmaceutically acceptable salts thereof to treat such disorder. 2.The method of claim 1, wherein the amount is 0.03 mg to 100 mg/kg/day.